A number of different high and low copy number vector systems using a diversity of regulable promoter systems have been successfully developed to manipulate gene expression in Gram negative organisms such as Escherichia coli. As a result, E. coli can be genetically manipulated in a number of ways that have lead to a thorough understanding of the molecular basis for gene expression and to the elucidation of the function of many important proteins. As a further result, E. coli has been used as a production organism for the high level expression of a number of protein products, some of which are toxic. The majority of these vector systems developed in E. coli, however, do not function properly in Gram-positive microorganisms, likely due to physiological differences between Gram-positive and Gram-negative species (de Vos, W. M., et al., Curr. Opin. Biotechnol. 8:547-553, 1997; de Vos, W. M., and G. F. M. Simons, xe2x80x9cGene cloning and expression systems in lactococci,xe2x80x9d pp. 52-105. In M. J. Gasson and W. M. de Vos (ed.) Genetics and Biotechnology of Lactic Acid Bacteria. Routledge, Chapman and Hall Inc., New York, N.Y., 1994). The lack of vectors providing for the efficiently regulated expression of genes in Gram-positive bacteria has been responsible, in part, for the lack of suitable Gram-positive systems for production of valuable gene products on an industrial scale.
The characterization of the biology of Gram-positive bacteria has been hampered by the lack of cloning and expression vector systems that are stably maintained, tightly regulated and inducible, analogous to those developed in E. coli. As a result, the study of important Gram-positive pathogens, that can cause a variety of different illnesses including life threatening ones, has been severely limited, impeding the discovery of novel, life saving therapies to treat infectious diseases.
In the last decade, the rapid rise in severe and fatal infections caused by drug resistant microbial pathogens has presented a significant threat to public health worldwide. One of the pathogens of immediate concern is the Gram-positive organism Staphylococcus aureus. There are over nine million cases of S. aureus infections a year. S. aureus infections are one of the most prevalent types of hospital acquired infections requiring treatment. Methicillin resistant S. aureus now represents a significant proportion of all Staphylococcus aureus infections in hospitals. Although these infections can be treated with the antibiotic vancomycin, it has been documented that S. aureus strains can become resistant to this last line antibiotic. There is genuine concern in the medical community that new antibiotics to treat S. aureus infections must be discovered in order to prevent a return to the preantibiotic era where death by bacterial infection was common.
A necessary first step towards the identification of novel therapeutics to treat Staphylococcus aureus and other Gram-positive bacterial infections is an ability to clone and regulate the expression of genes in the organism in order to understand its biology. To do this, repliconsxe2x80x94replicating units of DNA or RNA, such as plasmidsxe2x80x94should be constructed to carry the genes and ensure the desired level of gene expression when the replicons are in the bacterial cells. A system using specially constructed bacterial strains is needed to control and evaluate expression in vitro as well as in vivo during a bacterial infection in an animal model system so that the biology of the infectious disease process can be investigated.
To provide broad practical applications of a Gram-positive inducible gene expression system, it is desirable for the system to have the following features: (1) The system is under tight expression control that avoids or minimizes leaky expression of a cloned gene. Leaky expression can often result in cell toxicity and so must be avoided. (2) The system responds with high levels of expression upon induction. (3) The inducibility of the system is independent of growth media so that a variety of environmental conditions can be evaluated. (4) Administering inducer to the gene expression system, by itself (that is, without production in the cells of the regulated gene product) does not cause significant change in the phenotype of the bacteria (e.g., inhibit the growth of the bacteria). (5) The system functions with features (1), (2), (3) and (4) not only when the bacteria are grown in culture, but also in an animal infection model. To date, there has been no acceptable inducible expression system that covers the above listed criteria. In addition, if improved cloning and expression systems were available a wealth of opportunities could be realized for the efficient and economic utilization of microorganisms for the industrial production of macromolecules.
The invention encompasses a replicon which can be used for the high-level, inducible production of a gene product in Gram-positive bacteria, including, but not limited to staphylococci (for instance, S. aureus and S. carnosus), and Gram-positive bacilli (for instance, Bacillus subtilis). The invention encompasses a replicon comprising a promoter/operator region that causes gene expression under its control to be tightly repressed in the absence of tetracycline or an analog of tetracycline. However, in the presence of tetracycline or an analog thereof, the promoter/operator region causes a high level of gene expression of a gene situated downstream of the promoter. That is, the promoter/operator can produce a high level of transcription, but is tightly regulated (non-leaky).
Built into the replicon is a linker site downstream of the promoter/operator region so that insertion of a segment of nucleic acid encoding a gene product (wherein the segment comprises an open reading frame) at the linker site can put the transcription of such an open reading frame under the control of the promoter/operator region. Optionally, a linker site can be within or adjacent to a segment of nucleic acid encoding a carrier polypeptide, such that insertion, in frame, of a nucleic acid segment encoding a second polypeptide (which can be as short as a few amino acid residues, and usually termed a xe2x80x9cpeptide,xe2x80x9d but here, included in the term xe2x80x9cpolypeptidexe2x80x9d) results in a derivative replicon, which when introduced into an appropriate host species of bacteria, can inducibly produce a fusion polypeptide (also, xe2x80x9cfusion proteinxe2x80x9d) of the carrier and second polypeptides.
A particular promoter/operator region, called PJT/TetO herein, and which has been described herein as a part of pC3875, has been found to be particularly well suited to the tightly controlled tetracycline-regulated expression of genes inserted downstream from the promoter/operator. (Herein, xe2x80x9ctetracycline-induciblexe2x80x9d or xe2x80x9ctetracycline-regulatedxe2x80x9d describes genetic elements responsive to tetracycline as well as to analogs thereof that act similarly to tetracycline.)
Also an aspect of the invention is a strain of Gram-positive bacteria comprising one or more genes encoding tetracycline resistance [tet(M), tet (0), etc.] and tetR (tet repressor) genes. Construction of such a strain of S. aureus is described herein.
A further aspect of the invention is a system for inducible expression of a gene, comprising Gram-positive bacteria bearing a replicon, wherein the replicon comprises a tetracycline-inducible promoter/operator region for the tightly regulated control of gene expression, for example, the promoter/operator PJT/TetO (for tet operator, originally described in transposon Tn10, see Wissmann, A. et al., J. Mol. Biol. 101:397-406, 1988; also Geissendorfer, M. and W. Hillen, Appl. Microbiol. Biotechnol. 33:657-663, 1990), and further comprising an open reading frame downstream of said promoter/operator region, wherein the open reading frame can be a coding sequence for a polypeptide, which can be a fusion polypeptide, for example. The Gram-positive bacteria can be S. aureus, for instance.
The replicon of the gene expression system can be constructed to have, instead of an open reading frame immediately downstream of the promoter/operator region, a linker site for the insertion of a segment of nucleic acid comprising a coding region for a gene of interest. The replicon of the gene expression system can also be constructed to have a linker site downstream of the promoter/operator region, wherein the linker is within or adjacent to a coding region for a carrier polypeptide, so that insertion of a second coding sequence results in tetracycline-inducible production of a xe2x80x9ccarrier-secondxe2x80x9d fusion polypeptide in cells bearing the replicon.
The system of gene expression described herein is especially well suited to the production of and/or analysis of the effects of gene products that may be toxic to the host cells. The system has further advantages in being suitable for use in an animal testing method. In this method, animals are infected with a strain of engineered bacteria, such as S. aureus (herein, the strain of engineered bacteria is an xe2x80x9cinducible system for expression of a genexe2x80x9d) where the inducible gene encodes a xe2x80x9ccarrier-peptidexe2x80x9d fusion protein. The peptide portion of the fusion protein encoded by the bacterial replicon is a candidate for causing a phenotypic effect on the host bacterial cells, typically, by enhancement or inhibition of the function of a host cell component, such as the activity of a host bacterial cell enzyme, resulting in a slowing or cessation of growth of the host bacterial cells. Tetracycline or inducing derivatives or analogs thereof can be safely given to the infected animals, to induce the controlled production of the xe2x80x9ccarrier-peptidexe2x80x9d fusion protein. If the tetracycline induction of gene expression of the xe2x80x9ccarrier-peptidexe2x80x9d fusion protein produces a slowing or cessation of growth of bacterial cells infecting the test animals (resulting in animal survival), then the peptide portion of the fusion protein is proven effective as having antibiotic action. It can be concluded then, also, that the peptide portion is affecting a target cell component that is essential to growth of the bacterial cells. Further measures can be taken to find more physiologically stable structural analogs of the peptide or to otherwise develop antibiotics modeled on the structure of the peptide.